SOME ASPECTS OF CHEMICAL CHANGES DURING
INSECT METAMORPHOSIS
BY ALTERBURY COULSTON EVANS.
(From the Department of Physiology and Biochemistry,
University College, London.)
(Received 23rd January, 1932.)
(With Five Text-figures.)
INTRODUCTION.
THE complex chemical changes associated with metamorphosis have long been of
interest to biochemists, but a detailed study of one type is not yet available, in spite
of much attention having been paid to the silkworm.
The subject of this study is the sheep blow-fly Lucilia sericata Meigan. This
insect is very suitable for an investigation of this kind since the prepupal stage,
morphologically similar to the larval stage but physiologically belonging to the
pupal stage, can be isolated with ease from the larvae. Histolysis and histogenesis
are well marked.
The changes in protein and its degradation products, skeletal or chitin-N, total
carbohydrate and fatty acids are studied at frequent intervals during the metamorphosis from larva to adult.
ANALYTICAL METHODS.
Material. The larvae were bred in an outdoor insectory and when full-grown
were transferred to clean, dry sand in which to pupate and kept in the laboratory
under constant-temperature conditions. The batch on which the changes in nitrogen distribution were studied was kept at 220 C, while the batches on which
changes in carbohydrate and fatty acid content were studied were kept at 170 C.
Nitrogen distribution. The malpighian tubules and that portion of the alimentary
canal containing excreted matter were dissected out from 5 gm. of material. The
remainder was ground to a paste and then extracted and filtered three times with
20 c.c. of distilled water. The remaining material, less a correction for the chitin
present, was taken to represent the insoluble protein which was regarded as an
approximation to the amount of organized tissue present. The filtrate was made up
to 70 c.c. and 30 c.c. of 10 per cent, trichloracetic acid added. This precipitated
quantitatively the soluble proteins, leaving proteoses, etc., in solution. These soluble
proteins were regarded as consisting chiefly of soluble reserve proteins and products
of histolysis. After removing the soluble proteins on a Buchner filter, the filtrate
Some Aspects of Chemical Changes during Insect Metamorphosis 315
was made up to 100 c.c. and a Serensen amino-acid titration carried out on two
25 c.c. portions. The proteoses and peptones in the remaining 50 c.c. were precipitated by adding 7 c.c. of o-66 iV sulphuric acid and 7 c.c. of 10 per cent, sodium
tungstate, giving a final concentration of about 1 per cent, tungstic acid. The total
nitrogen remaining in the filtrate together with the dissected material was estimated.
This nitrogen represented amino-N for which a correction could be made, excretory-N and the non-amino-N present in the amino acids. Unfortunately the two
latter cannot be separated and the figures given here for excretory-N represent
their sum.
Total carbohydrate. About 2-5 gm. of material were hydrolysed in 5 per cent.
HC1 for 4 hours. 5 c.c. of 20 per cent, phosphotungstic acid in 5 per cent, sulphuric
acid were added to precipitate reducing substances other than glucose. The excess
acid was removed from the filtrate with barium hydrate. The glucose was estimated
by the Hagedorn-Jensen method.
Fatty acids. 5-0 gm. of material were sufficient for this estimation. The soap
solution obtained by saponifying the tissue in 10 per cent, aqueous KOH for 3 hours
was thoroughly washed with ether to extract ether-soluble unsaponifiable matter
and the fatty acids recovered by acidifying the soap solution. The recovered acids
were resaponified and again washed with ether to ensure the absence of unsaponifiable matter. The recovered acids were weighed.
The proportions of saturated and unsaturated acids present were estimated by
two independent methods:
(a) By taking the iodine and thiocyanogen values and calculating the percentage
of saturated and unsaturated fatty acids present (Mitchelldo)).
(b) By Hilditch's modification of Bertram's oxidation method (Hilditch and
Priestman(s)).
RESULTS.
Nitrogen distribution.
Many previous workers have demonstrated that no nitrogen is lost during metamorphosis, Heller (6), Inouye(7), Kellner, Sako and Swano(S), Tangl(ia). This conclusion is further supported by the results in Table I, no nitrogen being lost once
the larvae have completely evacuated their gut.
Table I. Nitrogen distribution in milligrammes per 100 individuals
reared at 22° C.
Day
Insoluble protein
Soluble protein
Peptone
Ammo acids
Excretory-N
Chitin-N
Total N
8
1
2
4
6
649
73-i
412
4-3
2-8
608
405
7-0
26
410
43 7
39-3
69
§•7
33
u-3
34-7
1369
34-7
34 7
34'7
I35-J
137-0
134-5
58
49
49
84
132 6
8
i8-4
179
1346
135-7
9-8
3'4
9
11
14
15
33 9
30-1
58-5
64-5
SS-6
437
92
47-7
183
S-6
31
136
2-9
145
V*
i-8
176
7-8
165
15-5
34-7
137-7
36-7
134-2
67
2'I
20-2
3
i6
ALTERBURY COULSTON EVANS
These figures show that total protein reaches its maximum on the second day
after cessation of feeding (Fig. i). The corresponding decrease in degradation products suggests that this completes the synthetic activities of the larval stage. Histolysis now sets in and the total protein falls. This fall mainly concerns the insoluble
protein which falls rapidly until the ninth day. The insoluble protein which is
broken down during the prepupal stage would appear to be mobilized specially
for the production of chitin to form the strong puparium which is secreted at pupation. The soluble protein shows a slight decrease until pupation, after which it
increases at the expense of the still decreasing insoluble protein. Certain of the
imaginal organs have been developing during this time, notably the primordia of
the trophi, legs and wings, but the histolysis of fat body and certain muscles completely masks this in the data obtained.
vidi
•a
•3
g
0
o
HO
COtnUMIKti
70
•
60
SO
40
I
I
1
•
* " " * "
—
-
^
**
V
30
20
••sa
10
•
ee
6
8
10
12
14
15
Days at 22°C.
Fig. i. Curves showing the distribution of insoluble
protein-N
; soluble protein-N
.
Peptones increase to a maximum on the sixth day when pupation occurs and
then decline until the eleventh day when they slowly increase until emergence
(Fig. z). If the amount of peptone present were only related to the degradation of
protein, we would not expect a decline to occur after the sixth day, but rather that
it would remain constant or even increase while protein degradation continued.
This evidence suggests that an adequate supply of peptone is specially produced for
utilisation when chitin synthesis is occurring.
The concentration of amino acids increases until pupation occurs, remains
constant until histogenesis begins, and then slowly falls until the fly emerges. This
is in conformity with the work of Heller (6) and Courtois oo on certain Lepidoptera.
The formation of excretory nitrogen is most rapid from the fourth until the
ninth day and apparently ceases a few days before emergence.
Although histogenesis is occurring during the early stages of metamorphosis, it
does not become predominant over histolysis until about the tenth day when the
imaginal muscles begin to form. Growth of muscle is especially noticeable between
the tenth and twelth days, when the large thoracic muscles are formed. During this
Some Aspects of Chemical Changes during Insect Metamorphosis
317
period the insoluble protein increases very rapidly at the expense of the soluble
protein.
This study of the nitrogen distribution presents, in broad outline, a picture of
the tissue changes occurring during metamorphosis, first a mobilisation of protein
to form the hard protective puparium, then further degeneration of the larval
tissues which, with reserve material, are built up into the tissues of the adult.
c 20
a
p
S 10
I
2
4
6
8
14
10
16
Days at 22° C.
Fig. 2. Curves showing the distribution of chitin-N amino-N. —• —; excretory-N. ..
; peptone-N.
;
Carbohydrate.
Glycogen has been found in this species, contrary to the finding of Frew (4),
on "blow-flies" (species not stated, but probably Calliphora sp.). WeinIand(i4) reports a small amount to be present in Calliphora and Claude Bernard (O
states that large amounts are present in " asticots." In attempts to isolate glycogen,
a white non-reducing substance was obtained, insoluble in 70 per cent, alcohol,
giving an opalescent solution in water, a reddish colour with iodine which did not
return after boiling. On boiling this material with dilute HC1 no reduction was
obtained with Fehling's or Benedict's solutions or by Cole's picric acid method.
The substance showed no reactions characteristic of protein or protein degradation
products. On further examination this may explain the apparent contradiction
expressed above.
The amount of glucose falls steadily during histolysis until the thirteenth day
and then remains fairly constant during histogenesis (Fig. 3).
Table I I . Milligrammes of glucose per 100 individuals reared at iy° C.
(Duplicate estimations on one batch.)
Days
A.
B.
1
3
77-1
83-5
676
S
511
13
16
19
22
2O - 2
13-5
16-0
2O-2
158
10-3
19-1
33-5
7
10
4&-S
44-5
129
ALTERBURY COULSTON EVANS
Fat.
Previous work on the metabolism of fat during the metamorphosis has been
mainly concerned with ether-soluble material, In this study attention has been
focussed on the changes in fatty acid content.
Vaney and Maignonto), Kotake and Sera(o), and CouvreurO) have studied the
utilisation of ether-soluble material during the pupal stage of the silkworm. The
results of these investigations are in good agreement and show a rapid fall in the first
4-5 days, followed by a period of slight decrease. A somewhat similar state of
affairs exists in the worker bee according to Straus (n). In the blow-fly the fatty
acid content decreases rapidly immediately feeding has ceased (Fig. 4). This rapid
decrease continues until about the eighth day when it decreases more slowly. However, instead of continuing to decrease slowly a definite synthesis of fatty acid occurs,
and a second maximum is reached on the fourteenth day. Further decrease now
sets in and continues until emergence. This synthesis occurred in the three independent batches studied, two in detail and the third at three critical points.
80
r
60
40
20
2
4
6
10
8
12
14
16
18
20
22
Days at i7°C.
Fig. 3. The utilisation of carbohydrate.
Table III. Milligrammes offatty acids per ioo individuals reared at 170 C.
A.
Days
2
4
6
9
12
IS
17
19
2o8'2
166-1
II2'6
1277
205-0
194-6
161-5
1648
B.
Days
0
3
7
JO
13
17
2O
2393
l6o'2
113-8
74-5
122-4
9 2-2
60-5
The source of the synthesised fatty acids is of interest. Although there is a
diminution in the amount of carbohydrate during the period of fat synthesis, it is
Some Aspects of Chemical Changes during Insect Metamorphosis 319
not sufficient to account for the amount synthesised. As the respiratory quotient
during this period does not rise above 0-73, the idea that carbohydrate is the source
is further discredited. No proof that protein is the sole source of this fat exists, as
only about half of its carbon content can be accounted for from this source. The
fatty acid and excretory-N estimations were carried out on two separate batches of
larvae, reared at different temperatures and other varying conditions may have
helped to complicate matters. Weinland (14) has suggested that fat may be synthesised
from protein by Calliphora.
In order to study in more detail these interesting changes in fatty acid content,
the iodine and thiocyanogen values of the fatty acids obtained from one detailed
study were taken and the conclusions as to the composition of the fatty acids arrived
at from these were confirmed by estimating the percentage of saturated fatty acids
present at three critical points by Hilditch's modification of Bertram's oxidation
250
200
•a-g 150
c ^ 100
bo
60
2
A
6
8
10
12
14 16
18
20 22
Days at I7°C.
Fig. 4. The utilisation of total fatty acids.
method. The results, which are in good agreement (Tables IV and V), show that the
amount of saturated fatty acids present decreases very slightly, if at all, and the
changes in the fatty acid content chiefly concern the unsaturated acids, which decrease rapidly until the tenth day. A synthesis of unsaturated fatty acids only now
occurs, reaching a maximum on the thirteenth and fourteenth days. A further
decrease sets in and continues until emergence (Fig. 5).
Table IV. Milligrammes of saturated fatty acids per 100 individuals.
DayB
By calculation from iodine and
thiocyanogen noa.
Hilditch's method
0
3
7
33"5
276
27-5
38-3
10
156
13
17
189
21-7
20 0
320
ALTERBURY COULSTON EVANS
Table V. Milligrammes of unsaturated fatty acids per ioo individuals.
Days
By calculation from iodine and
thiocyanogen nos.
Hilditch's method
o
3
7
205-8
132 6
86-3
10
13
i°3-5
58-8
2009
17
77 9
IO2'4
260
200
•tf-g 'so
•S3
| |
I
100
60
2
4
8
8
10
12
14
16
18
20
Days at 17° C.
. Unsaturated
Fig. 5. Analysis of Fig. 4. Unsaturated acids. Iodine and Thiocyanogen values,
acids. Hilditch's modification of Bertram's method, - - -. Saturated acids. Iodine and Thiocyanogen
. Saturated acids. Hilditch's modification of Bertram's method,
values,
During the initial fall, the acetyl value of the fatty acids is o-o showing that no
conversion of unsaturated fatty acids into hydroxy acids is taking place. The respiratory quotient during the period is 0-70, indicating a complete oxidation of the fatty
acid molecule.
Characterisation of the fatty acids.
Mean molecular weight
Iodine value...
Thiocyanogen value
Acetyl value
Melting points
Solidification points
270-287
79-96
69-78
o-o
23-33° C
21-25° C
SUMMARY.
The changes in carbohydrate content, fatty acid content, and nitrogen distribution are described in detail during the metamorphosis from larva to adult in the
sheep blow-fly, Lucilia sericata Meigan.
Carbohydrate decreases rapidly until the thirteenth day, after which it remains
constant. Glycogen is present.
Some Aspects of Chemical Changes during Insect Metamorphosis 321
The fatty acid content decreases rapidly until the ninth day. A synthesis of
fatty acid now occurs, reaching a maximum on the fourteenth day, after which a
decrease sets in until emergence of the adult. The amount of saturated fatty acid
present remains constant, the unsaturated fatty acids only being utilised.
The course of histolysis and histogenesis is reflected in the nitrogen distribution
curves. The initial decrease in insoluble protein correlated with a rise in peptone is
associated with the formation of the hard chitinous puparium. Continued decrease
of the insoluble protein accompanied by a fall in peptone and an increase of soluble
protein continues until histogenesis of the imaginal thoracic muscles commences.
The insoluble protein now abruptly rises and the soluble protein shows a corresponding decrease. Formation of excretory-N occurs mainly during histolysis.
The larvae used for this study were bred at the London School of Hygiene and
Tropical Medicine by kind permission of Dr P. A. Buxton. I am greatly indebted
to Dr R. P. Hobson for his kindness in rearing the large number of larvae used.
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(3)
(4)
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